PROJECTSUMMARY Over the last few decades, the medical community has witnessed a remarkable shift in the composition of pharmaceutical therapies from traditional small molecules to biomacromolecules. Despite their tremendous success, macromolecular therapies have been limited almost exclusively to extracellular targets due to the significant challenge of their controllable delivery into the cytoplasm. To overcome this critical barrier in expandingthescopeofmacromolecuartherapy,naturallyoccurringself-deliverableproteinsystemssuchasthe Botulinum neurotoxins (BoNTs) offer an attractive solution. These protein complexes recognize cholinergic neurons,andpromotethetranslocationoftheactivetoxincomponent,theBoNTLCprotease,intothecytosol whereitdeactivatesthecellularmachineryresponsibleformembranefusioneventsthroughproteolysis.While potent,BoNTLCproteasesarehighlyspecificcatalysts,andgenerallyonlyrecognizeasinglesubstrate.Efforts toreprogramtheactivityofBoNTLCproteaseshavemetwithlimitedsuccess,andtheinaccessibilityofnew intracellulartargetsforBoNTLCproteasesremainstheprimarybarrierinextendingBoNT-derivedtherapeutic strategies.Recently,phageassistedcontinuousevolution(PACE)hasemergedasapowerfultoolfortherapid evolutionofnovelactivity,andhasbeenappliedtotheexplorationofthemechanismsbywhichviralproteases developresistancetoinhibitors,andtoevolvingnewsubstrateactivitiesinviralproteases.Thisworkwillextend thePACEstrategytoreprogramtheBoNTFLCproteasetocleavenoveltargetsequences,andwillbeapplied to the intracellular proteolytic deactivation of the therapeutically relevant SNARE protein VAMP7. With this in mind, the goals of this proposal are: (1) adapt the current protease PACE platform for use with BoNT LC proteasesandevolveaBoNTLCproteasetoselectivelycleavetheSNAREproteinVAMP7;?(2)developaPACE negativeselectionandapplythisselectiontoevolveBoNTLCproteasesthatcleaveVAMP7withhighspecificity;? (3)characterizeindepththeactivity,specificity,andpotentialtherapeuticrelevanceoftheevolvedproteases. This work will provide a strong foundation upon which future catalytic intracellular protease therapies can be based.